PROJECT SUMMARY/ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is an almost uniformly lethal disease with a 5-year survival of only 6%. Given the tremendous current focus on the creation of new treatment modalities for pancreatic cancer, the development of imaging technologies that can safely, accurately, and unambiguously aid in the early diagnosis, staging, and treatment monitoring of the disease is a vitally important unmet clinical need. While antibodies have long been attractive vectors for the delivery of diagnostic and therapeutic radioisotopes to cancer cells, radioimmunoconjugates often give rise to a critical clinical complication: high radiation doses to non-target organs. In order to circumvent this obstacle, we have recently developed a methodology for pretargeted PET imaging that harnesses the rapid bioorthogonal cycloaddition between trans-cyclooctene (TCO) and tetrazine (Tz) to effectively combine the affinity and specificity of antibodies with the rapid pharmacokinetics of small molecules. This methodology not only distinguishes tumor tissue with very high contrast, but also produces only a fraction of the background radiation dose to healthy tissue compared to directly labeled antibodies. This proposal describes the development, preclinical validation, and clinical translation of a pretargeted strategy for the PET imaging of PDAC. We will employ the antibody 5B1, a fully human monoclonal antibody (mAb) which targets CA19.9, a well- established biomarker for pancreatic cancer. Specific Aim 1 (SA1), executed during Years 1-2 of the award period, will be focused on the design, synthesis, and pharmacokinetic evaluation of a library of 68Ga-labeled Tz radioligands with the overall goal of identifying four radioligands with the most favorable combination of stability, reactivity, and pharmacokinetics. Specific Aim 2 (SA2), executed during Years 2-3 of the award period, will be centered on the in vivo evaluation of pretargeted PET imaging strategies employing these Tz radioligands and 5B1-TCO in three different murine models of PDAC. The different methodologies will be evaluated based on tumoral uptake, tumor-to-background activity ratios, and dosimetry metrics, and the overarching goal of this specific aim is the identification of a strategy suitable for clinical translation. Specific Aim 3 (SA3), executed during Years 3-5 of the award period, will be focused on the clinical translation of a pretargeted PET imaging strategy. To this end, an FDA IND will be prepared and submitted for the first-in- human clinical trial, and the in vivo performance of the methodology will be evaluated in a series of 28 patients to obtain preliminary imaging data and fundamental biodistribution, metabolism, and safety information. We believe that this proposal could have a significant near-term impact on the clinical care of patients with pancreatic cancer by creating a sensitive, safe, and effective diagnostic and theranostic imaging modality. Furthermore, we contend that this work could also have a long-term, transformational effect on the way antibodies are used in nuclear medicine, ultimately improving the imaging of a wide field of malignant growths.